Registration Dossier

Administrative data

Description of key information

Due to the high proportion of Bisphenol AF in the registered substance and the histopathological effects on male reproductive organs seen in the 28 -day repeated dose toxicity study in rats, classification as STOT RE 2, H373, with prostate and seminal vesicles as target organs, is warranted.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
August 2014 - August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
The deviation were considered not to affect the scientific integrity of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
The deviation were considered not to affect the scientific integrity of the study.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 37060B
- Expiration date of the lot/batch: 01 March 2019
- Purity test date: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han™:RccHan™:WIST strain rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: approximately six to eight weeks old.
- Weight at study initiation: the males weighed 199 to 217g, the females weighed 157 to 178g
- Fasting period before study:
- Housing: The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet and Water:The animals were allowed free access to food and water
- Acclimation period: The animals were acclimatized for six days during which time their health status was assessed

DETAILS OF FOOD AND WATER QUALITY: The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness

IN-LIFE DATES: The in-life phase of the study was conducted between 09 September 2014 (first day of treatment) and 07 October 2014 (final day of necropsy).
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily, for up to twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe.
Vehicle:
arachis oil
Details on oral exposure:
the test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined. Results show the formulations to be stable for at least eight days when stored at approximately 4 ºC in the dark.
Formulations were therefore prepared weekly during the treatment period, divided into daily aliquots and stored as above before use.

Samples of each test item formulation were taken and analyzed for concentration of XA31.
Duration of treatment / exposure:
up to 28 consecutive days
Frequency of treatment:
The test item was administered daily,
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low concentration
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Medium concentration
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High concentration
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were based on the available data from a 7 Day range-finding study ). In this study, males given the testitem at 500 or 1000 mg/kg bw/day and females at all dose levels showed body weight
impairment that was associated with a reduction in food intake at the start of the dosing period.Subsequent recovery in body weight gain was apparent, however weight gain and foodconsumption for males receiving the high dose and females at all dose levels were still lower than controls towards the end of the dosing period. In the absence of any clinical signs or macroscopic findings, it is likely that these animals would have attained full recovery over time.
At all dose levels, animals of both sexes receiving the test item also showed a persistent and occasionally dose-related increase in daily water intake in comparison with controls. Taking the overall results into consideration, a high dose level of 1000 mg/kg bw/day was considered to be suitable for use in the present study together with 100 and 300 mg/kg bw/day as the low and intermediate dose levels, respectively. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
Positive control:
None
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
> Time schedule: immediately before dosing, up to thirty minutes post dosing and one after dosing.

BODY WEIGHT: Yes
> Time schedule for examinations: on Day 1 and at weekly intervals thereafter

FOOD CONSUMPTION AND FOOD EFFICIENCY: yes
> Food consumption was recorded for each cage group at weekly intervals throughout the study.
> Food conversion efficiency was calculated retrospectively.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
> Time schedule for examinations: daily for each cage group.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
>Time schedule for collection of blood: on all surviving animals from each test and control group at the end of the study (Day 28)
>Parameters checked were as follow:
- Hemoglobin (Hb)
- Erythrocyte count (RBC)
- Hematocrit (Hct)
- Erythrocyte indices: mean corpuscular hemoglobin (MCH), - mean corpuscular volume (MCV), - mean corpuscular hemoglobin concentration (MCHC)
- Total leukocyte count (WBC)
- Differential leukocyte count: neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono),, eosinophils (Eos), basophils (Bas)
- Platelet count (PLT)
- Reticulocyte count (Retic)
- Prothrombin time (CT)
- Activated partial thromboplastin time(APTT)


CLINICAL CHEMISTRY: Yes
> Time schedule for collection of blood: on all surviving animals from each test and control group at the end of the study (Day 28)
>Parameters checked were as follow:
Urea
Inorganic phosphorus (P)
Glucose
Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.)
Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation)
Creatinine (Creat)
Sodium (Na+)
Total cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-)
Bile acids
Calcium (Ca++)
Triglycerides


URINALYSIS: Yes
> Time schedule for collection of urine: Urinalytical investigations were performed on all test and control group animals during Week 4. Urine samples were collected overnight by housing the rats in metabolism cages.
> Parameters checked in table [No.3] were examined.
Volume
Protein
pH
Ketones
Glucose
Urubilinogen
Bilirubin
Blood
Specific gravity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and on Days 7, 14, 21 and 25, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on all
animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: no


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (see table) / No / Not specified
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. For urine volume and specific gravity, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Urine volume and specific gravity were statistically analyzed using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pairwise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical observations considered to be related to the toxicity of the test item.
Mortality:
no mortality observed
Description (incidence):
There were no clinical observations considered to be related to the toxicity of the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean body weight gains in males from all dose levels were generally lower than controls throughout the study in a dose-related manner resulting in lower overall body weight gains for these animals. An improvement was apparent in males from the 100 mg/kg bw/day dose group towards the end of the treatment period and the effect on body weight development in males from the 300 or 1000 mg/kg bw/day dose groups was considered to be adverse.
At 300 or 1000 mg/kg bw/day, females showed reduced body weight gains at the start of dosing in a dose-dependent manner. Thereafter, fluctuations were apparent at all dose levels (no dose dependence) and overall weight gains in these females were slightly lower than controls. Taking into consideration the overall results, the effect on body weight development in these females was deemed of no toxicological significance
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 300 or 1000 mg/kg bw/day, food consumption in animals of either sex was marginally
reduced during the first week of dosing (dose-related) when compared with controls. Thereafter,an improvement was apparent for most of these animals although food intake for the high dose males remained slightly reduced over the second week. Overall food intake in these animals was slightly lower than controls. Food efficiency in males from all dose groups was generally lower than controls in a dose-related manner with the last week of dosing for the low dose males being an exception. In comparison, treated females only showed a few instances of lower food efficiency following intergroup differences in body weight gains and/or food intake.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Gravimetric measurement of water consumption revealed episodes of moderate to marked increases in water intake in males treated with 1000 mg/kg bw/day throughout the study and this was considered to be of possible toxicological significance. Females from all dose groups and males from the intermediate dose group also showed instances of slightly increased water consumption in particular during the first week of dosing; however, these were deemed not to represent an adverse effect of treatment with the test item.
Ophthalmological findings:
not examined
Description (incidence and severity):
Hematology investigations revealed decreased group mean hemoglobin, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration in males and females given 1000 mg/kg bw/day with these females also showing lower hematocrit and mean corpuscular volume. The toxicological significance of these changes was deemed uncertain. Any other changes were deemed unlikely to be of any toxicological significance.
Description (incidence and severity):
Blood chemistry analysis showed increased plasma concentration of triglyceride in females treated with 300 or 1000 mg/kg bw/day. Some individual values were above the historical dataranges and considering the change was in the opposite direction in the corresponding males (without achieving statistical significance), the toxicological significance of this finding was equivocal. Any other intergroup differences may have been influenced by liver changes and variations in plasma markers and may well be indicative of fluctuations in metabolic processes, but they were considered not to represent an adverse effect of treatment with XA31.
Description (incidence and severity):
Urine samples from animals of either sex treated with 1000 mg/kg bw/day and females from the 300 mg/kg bw/day dose group were generally slightly more acidic than controls. Additionally, urine samples from one male and two females from the intermediate dose group and two females from the high dose group showed the presence of hemoglobin and in the view of adverse histopathology findings in the kidneys from the intermediate and high dose animals, this finding may be of toxicological significance
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioral assessment scores across the treated animals of either sex remained similar to the respective controls.
There were no treatment-related changes in functional performance at any dose level.
Sensory reactivity scores remained unaffected by treatment with the test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing period, group mean absolute and body weight-related liver weights in animals of both sexes given 1000 mg/kg bw/day, in particular the females, were statisticallysignificantly higher than control. In light of microscopic observations in the liver from these animals, this finding was considered to be of toxicological significance. A dose-related reduction in absolute and body weight-related prostate and seminal vesicles (including coagulating gland) weights was also apparent in males from all dose groups relative to controls. Taking into consideration the histopathology findings from these tissues, the effect at 300 or 1000 mg/kg bw/day could be of toxicological significance. Any other statistically significant intergroup differences were considered to be of no toxicological importance.
Description (incidence and severity):
Treatment-related macroscopic findings of possible toxicological significance included small prostate and seminal vesicles (including the coagulating gland) in males receiving XA31 at dose levels of 300 or 1000 mg/kg bw/day. One male from the high dose group was also observed with small testes and epididymides; the toxicological significance of this finding was deemed equivocal.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Accessory Sex Glands: Minimal to moderate diffuse atrophy of the seminal vesicles, coagulating glands and the prostate gland was present in males given 300 or 1000 mg/kg bw/day. This finding may be a consequence of detrimental effect on dietary intake and body weight development; however, this could not be established with certainty and as such it was considered to be of possible toxicological significance. This observation was not seen in males treated with
100 mg/kg bw/day.
Kidneys: Minimal to moderate single cell necrosis and minimal incidences of proteinaceouscasts were observed in males given 300 mg/kg bw/day and animals of either sex receiving 1000 mg/kg bw/day. Additionally, minimal to moderate incidences of basophilic tubules were apparent in animals of either sex treated with XA31 at all dose levels. Although basophilic tubules were not seen in the kidneys from control animals, minimal basophilic tubules, without other evidence of degeneration or necrosis, are often seen spontaneously in laboratory rats and it was, therefore, considered likely that the occurrence of this finding in animals from the 100 mg/kg bw/day dose group was not a consequence of administration of the test item.

Liver: Minimal or mild centrilobular hepatocytic hypertrophy was observed in both sexes receiving 300 or 1000 mg/kg bw/day and females given 100 mg/kg bw/day; minimal or mild basophilia of periportal hepatocytes was also present in most of these animals. At 1000 mg/kg bw/day, minimal bile duct hyperplasia was observed in all animals along with minimal single
cell necrosis of the bile duct epithelium in a male and increased hepatocellular mitoses in a female. The centrilobular hypertrophy and periportal basophilia of hepatocytes was considered most likely a consequence of enzyme induction and minor metabolic perturbation and therefore adaptive and non-adverse. When present with bile duct changes, however, it was considered as an adverse change.

Mammary Gland: Feminine morphology, tubular and ductal profiles with female-like characteristics, were present in segments of the mammary gland from males in all dose group including one control male. According to Lucas et al. (2007), ‘occasionally, ducts and tubules with morphologic characteristics similar to those observed in female rats are observed in male rats subjacent to normal male lobuloalveolar morphology and should not be diagnosed as
feminization’, and it is possible that this distribution occurred spontaneously. In males given 1000 mg/kg bw/day, however, some of the female-like ducts were present within the main part of the gland and in view of the findings observed in the accessory sex glands, this finding was considered as possibly test-item related and of possible toxicological significance in these males.

Ovaries: The mild increase in atretic follicles seen in one female treated with 1000 mg/kg bw/day was considered most likely to have arisen by chance.

Testes: Mild spermatid retention, minimal tubular atrophy and minimal atrophy of Leydig cells was observed in one male given 1000 mg/kg bw/day with minimal tubular atrophy on its own also seen in a control male. As with the microscopic findings in the accessory sex glands, this finding could also have resulted from the effects of the food consumption and body weight deficit, and was considered as of equivocal toxicological significance.
Thymus: Minimal lymphocyte depletion observed in two females each from the 300 or 1000 mg/kg bw/day dose groups was considered to be stress-related rather than a direct effect of treatment with XA31.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
water consumption and compound intake
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
female reproductive system
Organ:
mammary gland
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
dorsolateral prostate gland
seminal vesicle
ventral prostate gland
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
Under the test conditions, changes in body weight development, food intake, water consumption, hematology, blood chemistry and/or urinalysis parameters were observed at dose levels of 100, 300 and 1000 mg/kg bw/day after oral administration. Microscopic examination of the selected tissues revealed also treatment-related findings in the kidneys and liver of both sexes, the accessory sex glands and possibly the mammary gland and testes of males.
Based on these results, the No Observed Adverse Effect Level (NOAEL) is considered to be the dose level of 100 mg/kg bw/day for systemic toxicity.
Executive summary:

The objective of this study was to assess the toxic potential of the Reaction mass of Amino¨phosphonium salt and BisphenolAF (XA 31), in a 4 weeks oral study to Han Wistar rats according to OECD Guideline 407 and EC Method B10. The oral route of administration was chosen to simulate the conditions of potential human exposure.

A previous range-finding test was performed in order to determine the test dose to be performed in the definitive test. In this preliminary study of the repeated dose toxicity study 28 days, the oral administration of the Reaction mass of Amino¨phosphonium salt and BisphenolAF (XA 31) to three groups of each of three male and three female Han Wistar rats at dose levels of 250, 500 and 1000 mg/kg bw/day by gavage for seven consecutive days was well tolerated.

 In this study, the test item Reaction mass of Amino¨phosphonium salt and BisphenolAF (XA 31) was administered by gavage to three groups, each of five male and five Han Wistar rats, for up to twenty-eight consecutive days, at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Urinalysis was also performed for all animals during Week 4 of the treatment period. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no treatment-related unscheduled deaths during the study and no relevant clinical signs considered to be related to the XA 31 were observed.

There were no treatment-related changes in functional performance at any dose level and the sensory reactivity scores remained unaffected by treatment with the test item.

There were test-item related effects on body weight at 100, 300 and 1000 mg/kg bw/day, food consumption at 300 and 1000 mg/kg bw/day and water consumption at 1000 mg/kg bw/day.

The toxicological significance of the hematologic changes observed at 1000 mg/kg bw/day were deemed uncertain.

The toxicological significance of the blood chemistry changes observed at 300 and 1000 mg/kg bw/day were considered equivocal.

There were treatment-related macroscopic findings of possible toxicological significance included small prostate and seminal vesicles (including the coagulating gland) in males receiving XA31 at dose levels of 300 or 1000 mg/kg bw/day.

The body weight-related liver, prostate and seminal vesicles weights were statistically significantly higher than control at 300 or 1000 mg/kg bw/day.

Histopathology findings were observed on accessory sex glands, kidneys, liver and possibly the mammary gland and testes of males at dose level either 300 or1000 mg/kg bw/day.

Under the test conditions, the oral (gavage) administration of Reaction mass of Amino¨phosphonium salt and Bisphenol AF (XA 31) to an Wistar strain rats, at dose levels of 100, 300 and 1000 mg/kg bw/day, was associated with changes in body weight development, food intake, water consumption, hematology, blood chemistry and/or urinalysis parameters. Microscopic examination of the selected tissues revealed treatment-related findings in the kidneys and liver of both sexes, the accessory sex glands and possibly the mammary gland and testes of males.

Based on the in-life observations and histopathology findings, a dose level 100 mg/kg bw/day is considered to be a No Observed Adverse Effect Level (NOAEL) for systemic toxicity.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is an OECD test guideline (408) and is of high quality (Klimish score = 1)

Additional information

Justification for classification or non-classification

Due to the high proportion of Bisphenol AF in the registered substance and the histopathological effects on male reproductive organs seen in the 28 -day repeated dose toxicity study in rats, classification as STOT RE 2, H373, with prostate and seminal vesicles as target organs, is warranted.